Ischemic heart disease (IHD) is the number one cause of morbidity and mortality in the US. With an estimated 1.6 million new or recurrent myocardial infarctions, the total economic burden of IHD on our health care system is tremendous. Although conventional pharmacotherapy and surgical interventions often improve cardiac function and quality of life, many patients continue to develop refractory symptoms. Thus, the development of new therapeutic strategies is urgently needed. "Tissue engineering" can be broadly defined as the application of novel engineering methods toward understanding structure-function relationships in normal or pathological conditions and the development of biological substitutes to restore, maintain, or improve function. It is different from "cell therapy" which intends to improve the function of an injured tissue by simply injecting suspensions of isolated cells. To date, two of the main limitations have been the lack of available methodology for scaling up the number of cells for potential clinical application and the inability to track the functionality of either cells or tissues after transplantation. Thus, the application of nanotechnology for development of novel techniques that address both deficits will provide a new catalyst for the entire field of investigation. The aims of our R21 proposal are to (1) significantly enhance the in vitro and in vivo performance of differentiated cardiomyocytes by electromechanical conditioning in an integrated stretchable microelectrode array-bioreactor system and (2) to engineer safe, durable, and biodegradable nanostructured scaffold sheets that can improve survival of conditioned cardiomyocytes. Once validated, we will transition into the R33 phase in years 3-5. The goals are (1) to scale-up nanostructured sheets from a single cell layer to a large area, multi-cell layer tissue graft, (2) to perform small animal in vivo assessment using molecular imaging techniques comparing the durability of injected cells alone versus our nanostructured tissue graft, and (3) to demonstrate safety and efficacy of our nanostructured tissue graft in a pre-clinical large animal model. We are confident that these developments will contribute substantially and help merge the fields of nanotechnology, tissue engineering, and molecular imaging. Progress in the field of cell therapy has been hindered by the lack of long-term engraftment and inability to monitor cell fate reliably post transplantation. Tissue engineering, by delivering cells with electrical and mechanical conditioning coupled to a supporting nanostructured scaffold may be a more promising and logical alternative. Our proposal uses multi-layers of conditioned cardiomyocytes patterned on a nanostructured scaffold to form a tissue graft that will be more durable, biodegradable, and safe. Concurrently, the application of molecular imaging modalities in small and large animal models will help us validate their functionality in vivo. With a multi-disciplinary team approach, we are confident that these combined efforts will accelerate the translation of nanotechnology-based tissue engineering to the clinical arena in the future.